Microanalysis for detection of analyte molecules is routinely employed in various analytical, bio-analytical and clinical applications. It is desirable that such assays have high specificity, use small volumes of reagents and samples, are performed as rapidly as possible and have high-sensitivity.
Assays are optimized to comprise a specific number of steps of standardized duration, along with various reagents, rinsing liquids, and other solutions of well-defined volumes. Once an assay is optimized, it can be routinely performed using standard conditions. An optimized assay may be sold as a kit, which means that a user runs the assay using a well-defined protocol and is ensured of having results within the specifications of the assays. Alternatively, an optimized assay may be integrated to a clinical analyzer or to other automated instrumentation.
An important limitation with assay technologies is that they address very different applications and different users. Ideally, assays should have maximum flexibility with respect to the number of steps and volumes of sample and reagent. Ideal assays have a large number of independent tests zones for calibrations and reproducibility purposes, and the best possible sensitivity. The technology around the assay such as the signal reader, pipetting system and other peripherals in general, are preferred to be versatile, inexpensive and compact. In contrast, the assays for diagnostic applications should be as simple to use as possible.
Surface assays, which involve the accrual of analytes on a surface, are widely used because they are convenient and sensitive. The analyte from a sample is singled out and accumulated on the surface with the help of a receptor specific for the analyte allowing washing off the remaining sample and interfering molecules. A classic example of surface assays would be an immunoassay wherein following steps are involved:                a “capture” antibody is placed on a surface        the surface is exposed to the sample and the capture antibody binds to its specific analyte        the surface is rinsed to remove the sample and interfering molecules        a second antibody conjugated to a reporter molecule (dye, fluorophore, radioactive isotope, enzyme . . . ) is provided and binds to the captured analytes        the excess of detection antibody is removed with a washing step        the signal associated to the detection antibody is measured. This signal is related to the concentration of analyte in the sample.        
The assays thus consist of multiple steps where samples, rinsing fluids, and reagents are successively employed. Microfluidic surface assays either are set for too specific applications, or require some peripheral equipment.
The receptors on surfaces and analytes in solution can be of various chemical or biological nature, such as cells, cell surface receptors, peptides, pathogens, chemicals, pesticides, pollutants, metals, metallic complexes, proteins, enzymes, antibodies, and antigens. To be utilized in an assay, a receptor and an analyte need to have a specific binding interaction. Cells immobilized on surfaces can for example be used to screen for specific analytes in solution. Conversely, ligands immobilized on surfaces can be used to screen for specific types of cells present in a solution. The receptors and analytes are sometimes called receptors and ligands. Existing devices and methods for performing microfluidic surface assays either are set for too specific applications, or require some peripheral equipment.
The known technology without using peripheral equipment for surface assay is based on the principle of lateral flow. In a lateral flow assay, a sample is added at the extremity of a device and capillary forces move the sample across zones where reagents have been placed and reach a zone with test sites. FIG. 1 depicts such a device where a capillary pump (10) is connected to the flow channel (30) and the test site (20) is located on the flow channel (30) where the assay reaction takes place. The rate of flow of the fluid in the flow channel (30) is defined by the capillary pressure. The technology based on lateral flow assay has been developed for specific applications where only one aliquot of sample (blood sample) is added to the device. This technology is not flexible and is not suited for typical assays in biology where multiple solutions and reagents must be employed for the assay.
U.S. Pat. No. 6,271,040 B1 uses the lateral flow approach for point-of-care testing applications. In U.S. Pat. No. 6,271,040 B1, the flow of the fluid is delayed by forming a hydrophobic three-dimensional pressure barrier at a region where the fluid should delay flowing. It can be used only when reagents are predisposed on the flow path of the sample. The device is sealed and the flow characteristics are determined for only one type of diagnostic application. Moreover, the pressure barrier should be formed in three-dimensional and hydrophobic surface modification, the fabrication process of which is complicated.
Another approach as depicted by FIG. 2 is the use of membrane to provide the capillary pressure needed to move liquids. The membrane also serves as a substrate for the assay. This approach is commonly used for point-of-care testing such as for pregnancy testing. The hydrodynamic flow properties of membranes are limited and difficult to optimize making each application cumbersome to develop. The membranes have to be synthesized to have appropriate porosity and hydrophilicity, must be able to incorporate reagents, and must not promote the non specific deposition of analytes of reagents in unwanted locations, for example as disclosed in U.S. Pat. No. 6,455,001. The degree of miniaturization that can be achieved using microfabrication techniques is not accessible to technologies based on membranes. In case cells are to be analyzed it would be difficult to analyze or detect cells using membranes because membranes hinder the motion of cells and particles and behave like filters.
U.S. Pat. No. 6,901,963 discloses a microfluidic device utilizing a capillary phenomenon comprising a flow channel for flowing fluid, the flow channel being formed between a top substrate and a bottom substrate; a flow blocking surface for stopping a flow of the fluid in the flow channel temporarily; and a hump for delaying the flow formed in the line of continuity with the flow blocking surface. This device utilizes capillary pressure to flow the fluid or applies additional pressure from the outside to the fluid. The flow of fluid is delayed by a capillary pressure barrier, which is generated by an aspect ratio of the flow channel at the flow blocking surface and a flow delay angle between the flow blocking surface and the hump for delaying the flow. The delay time of the flow is adjusted delicately by adjusting the length of the hump. The flow channel is formed with the top and bottom substrates formed of hydrophilic materials, hydrophobic materials, and/or a combination thereof. This device requires precise configuration, particularly on selecting and coating the flow channel substrates.
Technologies that are more versatile however need peripheral equipment such as the microfluidic devices using electro-kinetic flow principles, which need high voltage power supplies or pumps. Microfluidic technologies using acceleration forces to move liquids inside microconduits are emerging but they require a spinning platform and controlling circuits.
Elastomers have been proposed to be used as a pump to provide external pressure to allow the flow of the liquids. The elastomer has to be degassed and its refilling by air creates a pressure that can be used to draw liquids inside a microchannel. This approach is limited by the possibility of having leaks that could supply air to the elastomer and does not seem applicable for varying the flow conditions of a liquid in microstructures.
Capillary systems have recently been used with chip receivers to detect analytes with picomolar sensitivity and sub-microliter volumes of sample (Cesaro-Tadic et. al. 2004 Lab-on-a-chip, 2004, in press). To reach such sensitivity and miniaturization, the assays need extensive optimization and careful control of the flow rates of the various solutions. The flow rates are controlled by a heating element on surface of a chip receiver where the chip is placed. Pumps need to be actuated simultaneously using heat. In addition to needing peripheral equipment, the user needs to be an expert in setting the proper flow rates for his assay by actuating the heating element timely and accurately. Further, these devices are fabricated in Silicon [Si], which is an expensive material for fabricating chips with large capillary pumps. The precipitation of salts and proteins from solution in small capillary pumps due to evaporation is also an associated problem.